DNA sequencing is a fundamental research tool with wide-ranging applications. A common approach to DNA sequencing involves the subcloning of a large DNA fragments as smaller, overlapping fragments, the sequences of which are subsequently determined using the dideoxynucleotide chain termination approach (Sanger and Coulson, Proc. Natl. Acad. Sci. USA 74: 5463 (1977)).
Subcloning, and the restriction mapping required to efficiently subclone fragments, is a time consuming and labor intensive process. However, given the limitations associated with the amount of sequence which can be determined from a single extension reaction, it is necessary to initiate new sequencing reactions at a distance of about every 300-400 base pairs along a fragment, the sequence of which is to be determined.
One alternative to the subcloning approach is described by Henikoff et al. in U.S. Pat. Nos. 4,843,003 and 4,889,799. More specifically, Henikoff et al. describe a method in which a vector containing a DNA sequence of interest is linearized by digestion at two restriction endonuclease recognition sites, one generating a 5' overhang and the other a blunt end or 3' overhang. Timed digestion with E. coli Exo III from the 5' overhang, followed by treatment with a single-strand-specific nuclease generates a nested array of deletions. Unfortunately, this technique also is limited by the need for conveniently located restriction endonuclease recognition sequences.
An alternative to the approach described above was outlined by Chang et al. (Gene 127: 95 (1993)). Chang et al. describe a method in which a single-stranded nick is introduced at a position adjacent to the site at which a DNA fragment having a sequence which is to be determined is inserted in a cloning vector. The nick in the DNA is then extended under controlled digestion conditions to produce a single-stranded gap. The single-stranded gap is then treated with a nuclease which specifically digests single-stranded DNA, thereby producing a deletion within the DNA sequence of interest.
Chang et al. specifically report that the single-stranded nick in the DNA of interest cannot be expanded by treatment with E. coli Exo III. Given the fact that Exo III is a well-understood, relatively inexpensive enzyme, Chang et al. note that this is an unfortunate finding (page 96, column 2). The development of protocols which would enable the use of Exo III in such a DNA sequencing strategy would represent an important improvement in the art.